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Application Note
0057 - Milk Thistle for Silymarius by HPLC
As published in The Handbook of Analytical Methods for Dietary Supplements
Botanical Name: Silybum marianum (L.) Gaertn
Common Names: Marys thistle
Parts of Plant Used: Dried fruits
Uses: As an antihepatotoxic drug to treat
alcoholic liver disease and drug-induced
liver disease.
Modes of Action:
Various clinical trials have been performed on milk thistle that have
demonstrated positive effects for indications such as cirrhosis and alcoholic
liver diseases, hepatitis, and psychotropic-drug-induced liver damage.
Silymarin-type avonolignans are the bioactive components and they showed
antioxidative, antibrotic, anti-inammatory, and antilipid activity as well as
peroxidative, immunomodulating, and liver-regenerating effects. Silymarin can be
used successfully in therapy to promote faster regeneration of diseased liver and
silybinin was found to stimulate the activity of the DNA-dependent RNA-polymerase I,
thus increasing the rate of synthesis of all cellular proteins. The increase of protein synthesis offers a good explanation
for the liver-regenerating power of milk thistle.
14
Chemical Markers:
Milk thistle seed contains a group of compounds that includes avonolignans, avonoids, sterols, and fatty acids.
5,6
Silymarins are the bioactive components in milk thistle and generally the milk thistle extract is expressed as silymarin
percentage. Silymarins correspond to a group of avonolignans and the major ones are silybin A, silybin B, isosilybin
A, isosilybin B, silychristin, and silydianin. Due to the complexities of these structures, the stereochemistry of these
compounds, especially the stereochemistry of silybin A and B, and isosilybin A and B was resolved only recently.
7,8

Silymarins are used as marker compounds for quality control of milk thistle extract in the U.S. market.

O
O
O
H O
O H O
O H
O H
O C H 3
O H
O
O
O
H O
O H O
O H
O H
O C H 3
O H
Silybin A Silybin B

O H O
O H O
O H
O H
O
O H
O C H
3
O H
O H O
O H O
O H
O
O
O H
O H
C H
3
O
Silychristin Silydianin
Silybin A Silybin B Silychristin Silydianin
2 0057 - Milk Thistle for Silymarius by HPLC
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2011 ChromaDex, Inc. All rights reserved.
Methods of Analysis
The silymarin content in milk thistle has been analyzed by various methods including TLC, HPLC, UVvisible
spectrophotometry, and capillary electrophoresis.
911
Currently, UV-visible spectrophotometric and HPLC methods are
the most accepted for quality control of milk thistle extract. Although many HPLC methods have been published, few
methods can separate silybin A from silybin B and isosilybin A from isosilybin B.
Method 1:
The method at www.chromadex.com was used. This method can be used to analyze seven compounds including
silychristin, silydianin, silybin A, silybin B, isosilybin A, isosilybin B, and taxifolin.
Sample Preparation:
Weigh 500 mg of milk thistle extract into a 100-mL volumetric ask, add 25 mL of acetonitrile, sonicate for 15
minutes, cool to room temperature, and dilute to volume. Pipette 10 mL of this solution into a 50-mL volumetric ask
and dilute to volume with acetonitrile.
Chromatography:
Column: Phenomenex Luna C18 (2), 5 m, 150 4.6 mm.
Mobile phase: Solvent A = methanolwaterphosphoric acid (30:70:0.1 vol/vol/vol), solvent B = methanolwater
phosphoric acid (70:30:0.1 vol/vol/vol).
Gradient: 100%A to 100%B in 30 minutes, then keep at 100%B for 5 minutes.
Flow rate: 1.0 mL/minute
Injection volume: 5 L
Detection wavelength: 288 nm
Validation Data:
Not available
Method 2:
The method at www.nsna.org was used.
Sample Preparation:
For fruits, accurately weigh about 10 g of nely ground milk thistle fruit. Transfer the powder into an extraction
thimble within a continuous-extraction apparatus (250-mL round-bottom ask containing about 100 mL of hexane).
Extract the powder for 4 hours and discard the extract. Transfer the defatted fruit powder to a continuous-extraction
apparatus (250-mL round-bottom ask containing about 90 mL of methanol). Extract the powder for 8 hours, cool

O
O
O
HO
OH O
OH
OH
OH
OCH
3
O
O
O
HO
OH O
OH
OH
OH
OCH
3

O
O
O
HO
OH O
OH
OH
OH
OCH
3
O
O
O
HO
OH O
OH
OH
OH
OCH
3
Isosilybin A Isosilybin B
3 0057 - Milk Thistle for Silymarius by HPLC
10005 Muirlands Blvd., Suite G, Irvine, CA 92618 | Tel: +1-949-419-0288
Fax: +1-949-419-0294 | sales@chromadex.com | www.chromadex.com
2011 ChromaDex, Inc. All rights reserved.
down, and transfer to a 100-mL volumetric ask and dilute to volume with methanol. Dilute 1:25 with methanol and
lter a portion into an HPLC vial.
For the extract, accurately weigh (0.1 mg) about 70 mg of extract into a 100-mL volumetric ask. Add 70 mL of
methanol and sonicate for 20 minutes. Cool and dilute to volume with methanol.
Chromatography:
Column: YMC-Pack ODS-A C18, 5 m, 4.6 150 mm or Phenomenex Luna C18 (2), 5 m, 150 4.6 mm.
Mobile phase: Solvent A = methanolwaterphosphoric acid [Mix 200 mL of methanol with 800 mL of water. Mix 5
mL of phosphoric acid into 995 mL of the methanolwater (20:80)], solvent B = methanolwaterphosphoric acid
[Mix 800 mL of methanol with 200 mL of water. Mix 5 mL of phosphoric acid into 995 mL of the methanolwater
(80:20)].
Gradient:
Validation Data:
Not available, but this method is known to be a validated method.
Time (minutes) %A %B
0 85 15
5 85 15
20 55 45
40 55 45
41 85 15
55 85 15
Flow rate: 1.0 mL/minute
Injection volume: 10 L
Column temperature: 40C
Detection wavelength: 288 nm
4 0057 - Milk Thistle for Silymarius by HPLC
10005 Muirlands Blvd., Suite G, Irvine, CA 92618 | Tel: +1-949-419-0288
Fax: +1-949-419-0294 | sales@chromadex.com | www.chromadex.com
2011 ChromaDex, Inc. All rights reserved.
References:
1. Saller R, Meier R, Brignoli R. The use of silymarin in the treatment of liver diseases. Drugs. 2001;61(14):203563.
2. Sonnenbichler J, Sonnenbichler I, Scalera F. Inuence of the avonolignan silybinin of milk thistle on hepatocytes and kidney cells. In:
Lawson L, Bauer R, eds. ACS symposium series 691: phytomedicines of Europe. Washington, DC: American Chemical Society;
1998:26377.
3. Luper S. A review of plants used in the treatment of liver disease: part 1. Altern Med Rev. 1998;3(6):41021.
4. Leng-Peschlow E. Properties and medical use of avonolignans (silymarin) from Silybum marianum. Phytother Res. 1996;10(Suppl.
1):S256.
5. Khafagy SM, Abdel Salam NA, Abdel HR, et al. Constituents of Silybum marianum (L.) Gaertn. (Compositae). Scientia Pharmaceutica.
1981;49(2):15761.
6. Yin Q, Wang S, Nan J, et al. Analysis of fatty acids in Silybum marianum oil by GC/MS. Yanbian Daxue Xuebao, Ziran Kexueban.
1998;24(4):268.
7. Lee DY, Liu Y. Molecular structure and stereochemistry of silybin A, silybin B, isosilybin A, and isosilybin B, isolated from Silybum
marianum (milk thistle). J Nat Prod. 2003;66(9):11714.
8. Kim NC, Graf TN, Sparacino CM, et al. Complete isolation and characterization of silybins and isosilybins from milk thistle (Silybum
marianum). Org Biomol Chem. 2003;1(10):16849.
9. Kvasnicka F, Biba B, Sevcik R, et al. Analysis of the active components of silymarin. J Chromatogr A. 2003;990(1-2):23945.
10. Bilia AR, Bergonzi MC, Gallori S, et al. Stability of the constituents of calendula, milk-thistle and passionower tinctures by LC-DAD and
LC-MS. J Pharm Biomed Anal. 2002;30(3):61324.
11. Quaglia MG, Bossu E, Donati E, et al. Determination of silymarine in the extract from the dried Silybum marianum fruits by high
performance liquid chromatography and capillary electrophoresis. J Pharm Biomed Anal. 1999;19(3-4):43542.
Representative HPLC Chromatogram of Milk Thistle Extract Run by Method 1.